XB-IMG-172745
Xenbase Image ID: 172745
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Fig. 3. Hnf1β is required for pancreas development in vitro and in vivo
(A) Morpholino-mediated knockdown of Hnf1β in pancreatic explants. In order to
demonstrate the specificity of the morpholino-effect, RNA for a hormone-inducible version of
Hnf1β (Hnf1b-GR) was co-injected and explants were treated with the GR inducer
dexamethasone (DEX) together with RA at the equivalent of gastrula stage. At the equivalent
of stages 31 and 39, total RNA was isolated from approximately 30 explants each condition
and subjected to RT-PCR. Detection was for endogenous (endo) and injected Hnf1β (inj.), as
well as for the marker genes indicated. The Hnf1β loss-of function phenotype and its rescue
was observed for four independent biological replicates. (B) 4-cell-stage embryos were
injected with RNA coding for β-galactosidase (glb1) and either Hnf1β-morpholino or a
control-morpholino. At stage 32, embryos from two independent biological replicates were
used for WMISH against Pdx1 and Ptf1a and a real-time PCR analysis for Pdx1, Ptf1a and
Insulin. The graph indicates the fold change of tested markers in relation to Odc (ornithine
decarboxylase). ctr, uninjected embryos. (C) 4-cell-stage embryos were injected with RNA
coding for β-galactosidase alone or in combination with Hnf1β -GR RNA. At gastrula stage,
embryos were treated with dexamethasone (DEX) to induce Hnf1β function. WMISH against
Pdx1 and Ptf1a at stage 32 is shown. Boxplots display the range of the area percentage of
Pdx1 and endodermal Ptf1a domains in the endoderm observed in embryos from two
independent biological replicates (see Fig. S7). By the use of ImageJ (https://imagej.net),
Pdx1 and Ptf1a positive areas were measured (orange dotted line) in ratio to the area of the
whole endoderm (green dotted line). Values above the upper whisker, which is set at 1.5 x
interquartile range above the third quartile, are indicated as maximum outliers (°). (P-values
in an unpaired Student´s t-test **<0.01, ***<0.001). Image published in: Gere-Becker MB et al. (2018) © 2018. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license
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